A crucial function of human saliva is to protect oral tissues from the destructive effects of microorganisms. Since the oral cavity is a major route of entry for foreign and sometimes harmful bacteria into the body, it is necessary to keep a balanced, healthy flora. The balance between bacterial aggregation, stimulation, and prevention of adherence and growth, partially determines the development and composition of the oral microbiota. Certain normal oral flora bacteria have pathogenic potential if their population growth exceeds the normal oral flora balance.
Dental plaque, which develops on tooth enamel, can cause inflammation of gingival tissues giving rise to gingivitis. Chronic gingivitis is associated with the accumulation at the gingival margin of suptagingival dental plaque. There is a shift in the microbial composition of plaque in gingivitis away from a streptococci-dominated flora towards higher levels of pathogenic species. Over time, gingival inflammation and constant exposure to pathogenic oral bacteria can cause periodontitis. This most severe form of oral infection causes deterioration of tooth supporting tissues.
Lactoferrin (LF) is an iron-binding glycoprotein found in several mammalian exocrine secretions, such as milk and saliva. LF binds two atoms of Fe3+ and thus limits the availability of this essential nutrient for bacteria thereby causing an indirect bacteriostatic effect (Cole et al, Microbial aspects of dental curies, pp. 359-373, Information Retrieval, Washington, D.C. 1976). The iron-free form of LF damages the outer membrane of gram-negative bacteria by altering membrane function and permeability. LF also contains sialic acid. Human and bovine milk, which have sialic acid-containing fragments of k-casein, exert an anti-bacterial effect by binding LF to the bacterial cell wall and inhibiting the microbial adhesion to surfaces.
Glycomacropeptide (GMP), or k-casein glycomacropeptide, is a peptide prepared by food grade enzymatic hydrolysis and fractionation. The powder usually contains 89% GMP protein. The product contains glycosylated GMP (N-acetylneuraminic acid commonly known as sialic acid) and non-glycosylated GMP. It is the sugar chains containing sialic acid that are crucial for binding to bacteria surfaces (Kawasaki, Biosci. Biotech. Biochem. 56:195-198 (1992)). Since these chains bind to the active sites on certain oral bacteria, GMP acts to inhibit bacterial growth by binding to them, thereby keeping growth phase numbers from increasing (Kawasaki, Biosci. Biotech. Biochem. 57:1214-1215 (1993)).
The present invention results from studies designed to assess the efficacy of LF and GMP as anti-microbial agents. The bacteria tested in these studies include Streptococcus mutans, Streptococcus pyogenes, Actinobacillus haemophilus, and Pseudomonas aeruginosa. S. mutans is an alpha-hemolytic (partial destruction of red blood cells (RBCs) and hemoglobin that produces a greenish discoloration of the blood agar plate), nonmotile, facultatively anaerobic, Gram-positive cocci. The oral streptococcus species is found in the mouth cavity and upper respiratory tract of humans. These organisms hydrolyze sucrose and form dental plaque, and, as a result, create an anaerobic environment ideal for fermentation. S. pyogenes is a beta-hemolytic (complete destruction of RBCs and hemoglobin and result in a clearing around the growth on a blood agar plate) nonmotile, encapsulated, facultatively anaerobic, Gram-positive cocci. This species is responsible for strep throat, impetigo, middle ear infections, mastoiditis, and an array of infections resulting from hematogenic dissemination of the organism. It resides in the nose, throat and skin and when it becomes attached to host cells, it releases toxins that cause inflammation. P. aeritginosa is an aerobic, highly motile, straight or slightly curved, Gram-negative rod. It is common in soil and water and finds its way into the host by digestion, inhalation or through openings in the skin. It secretes tissue-damaging enzymes and forms a biofilm on dentures causing oral infections in denture users. A. haemophilus is an anaerobic, nonmotile Gram-positive cocci bacterium that is the most commonly found pathogen in individuals that have certain forms of periodontal disease. It leads to swelling of the gum tissue and the formation of periodontal pockets. It is less likely to be found in healthy oral flora.
Although S. mutens, S. pyogenes, A. haemophilus and P. aeruginosa are widely distributed among humans, an increased number in oral flora causes sore throats, scarlet fever, dental caries, gingivitis, and periodontitis. When populations of these bacteria exceed the normal flora amount, they begin to denature proteins, killing host leukocytes, host molecules, and dissolving blood clots. The goal of the studies that resulted in the present invention was to determine whether specific concentration levels could be identified at which LF and GMP would act as natural anti-microbial agents for the maintenance of oral flora.